The tRNA m1G9 methyltransferase (Trm10) is a member of the SpoU-TrmD (SPOUT) superfamily of methyltransferases, and Trm10 homologs are widely conserved throughout Eukarya and Archaea. Despite possessing the trefoil knot characteristic of SPOUT enzymes, Trm10 does not share the same quaternary structure or key sequences with other members of the SPOUT family, suggesting a novel mechanism of catalysis. To investigate the mechanism of m1G9 methylation by Trm10, we performed a biochemical and kinetic analysis of Trm10 and variants with alterations in highly conserved residues, using crystal structures solved in the absence of tRNA as a guide. Here we demonstrate that a previously proposed general base residue (D210 in Saccharomyces cerevisiae Trm10) is not likely to play this suggested role in the chemistry of methylation. Instead, pH-rate analysis suggests that D210 and other conserved carboxylate-containing residues at the active site collaborate to establish an active site environment that promotes a single ionization that is required for catalysis. Moreover, Trm10 does not depend on a catalytic metal ion, further distinguishing it from the other known SPOUT m1G methyltransferase, TrmD. These results provide evidence for a non-canonical tRNA methyltransferase mechanism that characterizes the Trm10 enzyme family.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.